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Article

AaSlt2 Is Required for Vegetative Growth, Stress Adaption, Infection Structure Formation, and Virulence in Alternaria alternata

by
Qianqian Jiang
1,†,
Tiaolan Wang
1,2,†,
Yongcai Li
1,*,
Yang Bi
1,
Miao Zhang
1,
Xiaojing Wang
1 and
Dov B. Prusky
3
1
College of Food Science and Engineering, Gansu Agricultural University, Lanzhou 730070, China
2
College of Applied Technology, Gansu Agricultural University, Lanzhou 730070, China
3
Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, Rishon LeZion 7505101, Israel
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work and share first authorship.
J. Fungi 2024, 10(11), 774; https://doi.org/10.3390/jof10110774
Submission received: 16 October 2024 / Accepted: 6 November 2024 / Published: 7 November 2024
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Abstract

:
Slt2 is an important component of the Slt2-MAPK pathway and plays critical regulatory roles in growth, cell wall integrity, melanin biosynthesis, and pathogenicity of plant fungi. AaSlt2, an ortholog of the Saccharomyces cerevisiae Slt2 gene, was identified from A. alternata in this study, and its function was clarified by knockout of the gene. The ΔAaSlt2 strain of A. alternata was found to be defective in spore morphology, vegetative growth, and sporulation. Analysis of gene expression showed that expression of the AaSlt2 gene was significantly up-regulated during infection structure formation of A. alternata on hydrophobic and pear wax extract-coated surfaces. Further tests on onion epidermis confirmed that spore germination was reduced in the ΔAaSlt2 strain, together with decreased formation of appressorium and infection hyphae. Moreover, the ΔAaSlt2 strain was sensitive to cell wall inhibitors, and showed significantly reduced virulence on pear fruit. Furthermore, cell wall degradation enzyme (CWDE) activities, melanin accumulation, and toxin biosynthesis were significantly lower in the ΔAaSlt2 strain. Overall, the findings demonstrate the critical involvement of AaSlt2 in growth regulation, stress adaptation, infection structure formation, and virulence in A. alternata.

1. Introduction

Alternaria alternata is a latent pathogen responsible for black spot disease in pear [1], jujube [2], sweet cherry [3], and various other fruits and vegetables globally, resulting in significant economic losses. Furthermore, A. alternata can generate AK, ACT, and other poisons, posing a risk to human health [4]. The predominant strategy for managing black spot involves the application of chemical fungicides; nevertheless, this approach has resulted in drug resistance and pesticide residues, raising food safety concerns, and contributing to environmental issues [5]. Thus, a thorough comprehension of the response and recognition mechanisms on host surfaces, together with the subsequent pathogenic processes of plant fungus, is essential for formulating targeted disease management methods.
Mitogen-activated protein kinases (MAPK) are part of the serine/threonine protein kinase family, comprising MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK), and MAP kinase (MAPK) [6]. MAPK cascade pathways are extremely conserved among eukaryotes, typically activated by various environmental stimuli, subsequently transmitting signals to downstream kinases and transcription factors, ultimately regulating the transcription levels of pertinent genes [7]. In plant fungus, MAPK pathways govern growth, secondary metabolism, stress adaptation, and pathogenicity [8,9]. Cao et al. [10] demonstrated that MAPK cascades provide crucial regulatory functions in proliferation, sporulation, and pathogenicity in Cytospora chrysosperma. In Colletotrichum gloeosporioides, the disruption of CgMK1 resulted in the loss of the ability to generate appressoria [11]. It has been reported that five conservative MAPK signaling pathways in Saccharomyces cerevisiae are regulated by Hog1 (high osmolarity response), Slt2 (the cell wall repair and integrity), Smk1 (sporulation-specific mitogen-activated protein kinase), Fus3 (the pheromone response), and Kss1 (the pseudohyphal and invasive growth upon nutrient deprivation), which are involved in osmoregulation, cell wall integrity, conidial assembly, pheromone response, and mating, respectively [12]. Notably, only three MAPK signaling cascades-Fus3/Kss1-MAPK, Hog1-MAPK, and Slt2-MAPK-have been discovered in filamentous fungi. Extensive research has demonstrated that the Fus3/Kss1-MAPK pathway regulates reproduction, pathogenicity, and conidial development [13,14,15], while the Hog1-MAPK route is implicated in osmotic stress, conidial development, and the virulence of several pathogens [16,17]. Our prior research validated that the AaHog1-MAPK cascade pathway is implicated in growth, infection structures formation, melanin accumulation, and virulence by pharmacological and molecular biology techniques in A. alternata [18].
The Slt2-MAPK pathway, also known as the cell wall integrity (CWI)-MAPK pathway, is a crucial component of MAPK cascades, significantly influencing conidia, oxidative stress, cell wall integrity, and the pathogenicity of pathogens [19]. Slt2-MAPK is crucial for sustaining the cell wall integrity in Beauveria bassiana [20]; analogous regulatory models have also been documented in Phytophthora sojae [21] and Ganoderma lucidum [22]. Slt2 is a downstream component of the Slt2-MAPK signaling pathway and is intricately associated with growth, development, cell wall integrity, and virulence [22,23]. The deletion of the AflSlt2 gene in Aspergillus flavus led to diminished virulence and atypical infection hyphae [24]. Moreover, Spada et al. [25] used silencing methods to show that Bmp3 influenced the development and pathogenicity of Botrytis cinerea.
Pharmacological experiments utilizing the MAPK pathway-specific inhibitor SB203580 (pyridoimidazoles, a specific inhibitor of the MAPK pathway) have previously proven that MAPK cascade pathways are involved in the development, infection structure formation, and pathogenicity of A. alternata [26]; nevertheless, the precise molecular processes remain ambiguous. This present research elucidates the roles of AaSlt2 in the vegetative growth, conidia development, stress adaptation, infection structure formation, and virulence of A. alternata via the developed AaSlt2 deletion mutant and complemented strains. These findings will contribute to the theoretical foundation for enhancing better disease control techniques.

2. Materials and Methods

2.1. Fungal Strains and Growth Conditions

The A. alternata JT-03 strain was prepared as a spore suspension (105 spores/mL), combined with 80% glycerol (Solarbio, Beijing, China) (3:1, v/v), and preserved at −80 °C. The wild-type (WT) strain was cultured on potato dextrose agar (PDA) at 28 °C and then configured for conidial suspension (105 spores/mL).

2.2. Bioinformatics Analysis

The AaSlt2 (XP_018391030.1) in A. alternata JT-03 was identified from A. alternata (SRC1lrK2f, taxon: 5599) utilizing the blastp algorithm with Slt2 (NP_011895.1) from S. cerevisiae. Amino acid sequences of Slt2 in other fungi were obtained from the NCBI database, and amino acid sequence alignments were conducted using DNAMAN 6.0. Conserved domains are a series of functional regions in proteins that remain relatively unchanged during evolution. By identifying these conserved domains in protein sequences, the function and evolution of proteins can be better understood. We predicted the conserved domains of AaSlt2 using Conserved Domains (https://www.ncbi.nlm.nih.gov/Structure/cdd, accessed on 6 January 2021).

2.3. Deletion and Complementation of AaSlt2

The AaSlt2 knock-out vector was acquired by a homologous recombination method (Figure S1). The upstream and downstream portions (about 1 kb) of the AaSlt2 gene were amplified from WT genomic DNA using the primer pairs AaSlt2-up/down-F/R (Table S1), and subsequently cloned into pCHPH upstream and downstream of hph, respectively. The fusion cassette comprising the upstream and downstream flanking sequences along with hph was subsequently introduced into the A. alternata JT-03 by employing Agrobacterium tumefaciens-mediated transformation (AtMT). The transformants were selected from induction medium plates with 200 μM acetosyringone (Solarbio, Beijing, China) and validated using PDA plates with carbenicillin (Solarbio, Beijing, China) (500 μg/mL) and hygromycin B (Solarbio, Beijing, China) (250 μg/mL). Additionally, deletion strains (ΔAaSlt2) were identified by PCR (Figure S2) and qPCR (Figure S3) using the primers listed in Table S2, respectively.
To achieve complementation, the fragment encompassing the CDS sequences of AaSlt2 was PCR-amplified (Figure S4) utilizing cDNA from the WT with the primer pairs listed in the Table S3. Subsequently, it was included into the pC-NEO-NGFP vector. The recombinant plasmids were introduced into the ΔAaSlt2 strain using AtMT-mediated transformation. The selected transformants were confirmed using PDA plates supplemented with containing kanamycin sulfate (Solarbio, Beijing, China) (50 μg/mL) and G418 (Solarbio, Beijing, China) (250 μg/mL). Additionally, complementation strains (ΔAaSlt2-C) were discovered by GFP fluorescence detection (not displayed) and PCR. Table S4 shows the list of primers used.

2.4. Determination of Spore Morphology, Vegetative Growth and Sporulation

To examine spore morphology, 20 µL spore suspensions of WT, ΔAaSlt2, and ΔAaSlt2-C were incubated on a slide, and viewed under a microscope. To observe vegetative growth, 2 µL spore suspensions of WT, ΔAaSlt2, and ΔAaSlt2-C were cultured on PDA at 28 °C for 5 days, with subsequent assessment of colony morphology and diameter. To ascertain of sporulation, 2 µL spore suspensions of WT, ΔAaSlt2, and ΔAaSlt2-C were incubated on PDA at 28 °C for 5 days. They were then collected and resuspended in conidia and enumerated under a microscope using a hemocytometer. The experiment was performed independently three times.

2.5. Gene Expression Analysis

Treatment 1: hydrophobic film (Univ-bio, Shanghai, China) was spread on the slide; treatment 2: hydrophobic film was spread on the slide, and then pear wax was evenly coated on the hydrophobic film. First, 20 µL spore suspensions (105 spores/mL) of WT were incubated on hydrophobic and pear wax extract-coated hydrophobic film. After 2, 4, 6 and 8 h of incubation, the spore slurry was harvested, centrifuged, and precipitated for further RNA extraction. RNA and cDNA were acquired using the manufacturer’s protocol (Vazyme, Nanjing, China). The primer pairs were listed in Table S5. The gene relative expression level was calculated using the 2−ΔΔCT method following the methodology of Livak and Schmittgen [27].

2.6. Infection Structure Formation Assays

The onion epidermis experiments were conducted according to the methodology of Tang et al. [28]. The inner membrane of the onion epidermis was excised and sectioned into 20 × 20 mm squares, which were subsequently positioned on slides (intact onion epidermis). Pear wax, dissolved in chloroform, was uniformly applied to the inner membrane of the onion with an applicator (pear wax extract-coated onion epidermis). Subsequently, 20 µL spore suspension of WT, ΔAaSlt2, and ΔAaSlt2-C was applied to intact onion epidermis (θ1) and pear wax extract-coated onion epidermis (θ2). The rates of spore germination, appressorium formation, and infection hyphae formation were assessed after 2, 4, 6 and 8 h of incubation. The experiment was performed independently three times.

2.7. Stresses Adaption Assays

Spore suspension of WT, ΔAaSlt2, and ΔAaSlt2-C was prepared as previously described. To evaluate the response of ΔAaSlt2 strains to cell wall stress and oxidative stress, PDA medium was formulated with congo red (Solarbio, Beijing, China) (100 μM), sodium dodecyl sulfate (Solarbio, Beijing, China) (SDS) (0.01%) and H2O2 (Solarbio, Beijing, China) (3 mM). Then, 2 µL spore suspensions of WT, ΔAaSlt2, and ΔAaSlt2-C were correspondingly deposited in the media. PDA medium devoid of such additives was designated as control. Colony morphology and diameter were assessed for 5 days at 28 °C. The experiments were conducted three times.

2.8. Assays for Pathogenicity

Pear fruits were sanitized with 0.1% of sodium hypochlorite (Solarbio, Beijing, China) and rinsed with tap water. After the surplus water was removed, the pear fruits were punctured with sterilized nails at three equidistant points around the equator of each pear. Subsequently, 20 µL of spore suspension from WT, ΔAaSlt2, and ΔAaSlt2-C were injected into each wound, respectively. The pear fruits were placed in a plastic bag at 28 °C. Following incubation periods of 3, 5, 7, 9 and 11 days, the diameters of the lesions were measured. Each treatment consisted of nine pear fruits.

2.9. Assessment of Cell Wall Degrading Enzyme Activity

The activity of the cell wall degrading enzymes (CWDEs) was monitored using method by Jia et al. [29]. Preparation of crude enzyme solution: the mycelium of WT, ΔAaSlt2, and ΔAaSlt2-C was inoculated into 15 mL of PDB and incubated at 28 °C with shaking at 220 rpm for 4 days. Following filtration through four layers of sterile gauze and subsequent rinsing with sterile distilled water, the mycelium was transferred to a medium containing pectinase (Solarbio, Beijing, China) and cellulase (Solarbio, Beijing, China) for a duration of 0 to 9 days. Following incubation for 1, 3, 5, 7 and 9 days, the samples were filtered and centrifuged at 4 °C and 12,000 rpm.
Assessment of polygalacturonase (PG) and pectinmethylgalacturonase (PMG) enzyme activities: addition of 0.5 mL of crude enzyme solution and inactivated enzyme solution to a pretreatment mixture consisting of 50 mM acetic acid-sodium acetate buffer, 10 g/L PG (Solarbio, Beijing, China) and PMG (Solarbio, Beijing, China) substrate, followed by incubation in a 37 °C water bath for 5 min, and subsequently for 1 h at the same temperature. Next, 1.5 mL of 3,5-dinitrosalicylic acid (Solarbio, Beijing, China) was added promptly, followed by boiling for 5 min, and then rapidly cooled to room temperature. The absorbance was recorded at 540 nm. The enzymatic activity of PG and PMG were assessed by measuring the quantity of reducing sugar liberated during the enzymatic reaction. The enzyme activity unit is defined as the quantity of enzyme necessary to facilitate the liberation of 1 μg of reducing sugar from the substrate by 1 mg of enzyme protein per hour at 37 °C. The experiments were conducted three times. The equation for determining enzyme activity is as follows: Enzyme activity (U/mg) = (C × N × OD)/T, where C represents substrate concentration, N denotes substrate amount, OD indicates absorbance value, and T signifies time.
Assessment of Cx (cellulase) and β-glucosidase activity: After altering PG and PMG substrates with Cx (Solarbio, Beijing, China) and β-glucosidase (Solarbio, Beijing, China) substrates, the remainder of the operation was identical to the aforementioned steps.

2.10. Measurement of Melanin

The melanin content was measured according to the method of Zhang et al. [30]. For standard curve preparation, melanin standards were created at several concentrations (0, 10, 20, 30, 40, 50, 60 mg/L), and the absorbance values of these solutions at 400 nm were recorded to construct a standard curve. Spore suspensions (2 µL) from the WT, ΔAaSlt2, and ΔAaSlt2-C strains were grown on PDA, covered with sterile cellophane sheets for five days at 28 °C. Weighed 0.25 g of mycelium was boiled in 1 M NaOH (Solarbio, Beijing, China). After cooling, the mixture was filtered and pH adjusted to 2.0. Subsequently, the crude melanin solution was obtained using centrifugation.
To purify and measure the melanin, 5 mL of 7 M HCl (Solarbio, Beijing, China) was added to the crude solution and heated in a water bath for 2 h. Subsequent to cooling, the mixture was centrifuged at 10,000× g for 15 min, followed by dissolution of the precipitate in 1 M NaOH, adjusting the pH to 2 using 7 M HCl, and centrifugation again at 10,000× g for 15 min. The purified melanin was obtained by repeating these steps three times. Following the dissolution of the precipitate with NaOH, the absorbance of the solution at 400 nm was quantified using an ultraviolet spectrophotometer. NaOH (1 M) served as the control solution. The formula for determining melanin content was y = x + 0.111/0.791 (where x is the absorbance value).

2.11. Measurement of Toxins

First, 2 µL spore suspensions of WT, ΔAaSlt2, and ΔAaSlt2-C strains were cultured on PDA, covered with sterile cellophane sheets for 5 days at 28 °C. Subsequently, 0.5 g of mycelium was weighed as sample. Toxins were extracted using an acetonitrile-water (80:20 v/v) solution with 0.3% formic acid (Solarbio, Beijing, China) and subjected to agitation for 30 min at 150 rpm. Next, 0.25 g of 0.04 M MgSO4 (Solarbio, Beijing, China) was added, followed by shock, centrifugation, and filtration of the supernatant using a 0.22 μm filtration membrane. This was then analyzed using an HPLC system. The separation and qualitative analysis of altenuene (ALT) and tentoxin (TEN) were conducted utilizing an Agilent 1260 Q-TOF mass spectrometer (Agilent, Santa Clara, CA, USA) fitted with an electrospray ionization (ESI) source. The HPLC parameters pertained to the testing methodology established by Xu et al. [31]. ALT and TEN standard products (HPLC > 98.0%) were acquired from Pribolab (Qingdao, China).

2.12. Statistical Analysis

Statistical analysis was performed using analysis of variance (ANOVA) in SPSS 18.0, with significant differences reported as 95% confidence intervals utilizing Duncan’s multiple range test. Pearson correlations was used to quantify the relationships among factors. The data are presented as the mean ± standard deviation of triplicate measurements.

3. Results

3.1. Identification and Bioinformatics Analysis of AaSlt2

AaSlt2 was anticipated to encode proteins with 416 amino acid residues. The conserved domain analysis revealed that the AaSlt2-encoded protein possesses a characteristic STKc_MPK1 domain, which is part of the PKc_like superfamily (catalytic domain of protein kinases) (Figure S5). The PKc_like superfamily mostly consists of the catalytic domains of serine/threonine-specific and tyrosine-specific protein kinases. It also encompasses RIO kinases, which are characteristic serine protein kinases, aminoglycoside phosphotransferases, and choline kinases. These proteins catalyze the transfer of the gamma-phosphoryl group from ATP to hydroxyl groups in specific substrates such as serine, threonine, or tyrosine residues of proteins.
Amino acid sequence alignment revealed that AaSlt2 is identical to other fungal Slt2 proteins, possessing a ‘TEY’ phosphorylation motif and characteristic sequences of the Ser/Thr protein kinase ‘VlHRDLKPGNLLV’ (Figure 1). To explain the functions of AaSlt2 in A. alternata, deletion mutant (ΔAaSlt2) and complemented strains (ΔAaSlt2-C) were produced.

3.2. AaSlt2 Is Involved in Spore Morphology Development, Vegetative Growth and Sporulation of A. alternata

Microscopic examination indicated that the wild-type spore was elliptical and developed a germ tube at the terminal tip, while the ΔAaSlt2 spore was elongated and produced a germ tube from the lateral side. Moreover, ΔAaSlt2 exhibited a greater number of septa compared to the WT. The spores of the ΔAaSlt2-C strain were equivalent to those of the WT strain (Figure 2A). The deletion of AaSlt2 resulted in a substantial drop in vegetative growth after 5 days of induction, demonstrating a 35% reduction in colony diameter, relative to the WT. The colony color of ΔAaSlt2 was drastically reduced compared to the WT, but ΔAaSlt2-C restored normal growth morphology (Figure 2B,C). The sporulation of ΔAaSlt2 was decreased by 68.13% (p < 0.05) relative to the WT (Figure 2D). The results indicated that the deletion of AaSlt2 is non-lethal in A. alternata; however, it induced abnormalities in spore morphology, vegetative growth, and sporulation.

3.3. AaSlt2 Is Essential for Infection Structure Formation of A. alternata

3.3.1. The Expression of the AaSlt2 Gene Is Up-Regulated During Infection Structure Formation

qRT-PCR analysis indicated that the expression levels of AaSlt2 were considerably up-regulated during infection structure formation of A. alternata under pear wax extract-coated surface. The expression levels of AaSlt2 peaked at the germ tube elongation stage (4 h) on both hydrophobic and pear wax extract-coated surfaces, exhibiting increases of up to 21.24 and 54.98 times, respectively, compared to the spore germination stage (2 h). During the germ tube elongation stage (4 h), the expression levels of the AaSlt2 were elevated on the pear wax extract-coated surface, being 1.59 times greater than on the hydrophobic surface (Figure 3), suggesting that AaSlt2 is essential for pear wax stimulated infection structure formation of A. alternata.

3.3.2. AaSlt2 Regulates the Infection Structure Formation of A. alternata on Onion Epidermis

The process of infection structural differentiation of A. alternata consists of four stages. Firstly, the spore germination extends the germ tube, which continues to elongate at the tip to form an appressorium. Over time, the tip of the appressorium extends into a bacterial filament (infection hyphae), which will infests the host, causing infection. To understand the roles of AaSlt2 in the infection structure formation of A. alternata, onion epidermis was used to simulate pear peel. The results indicated that pear wax extract-coated onion epidermis (θ2) markedly improved spore germination, appressorium formation, and infection hyphae formation in the WT, ΔAaSlt2, and ΔAaSlt2-C strains compared to intact onion epidermis (θ1). At the spore germination stage (2 h), more than 74% of the ΔAaSlt2 conidia did not germinate on pear wax extract-coated onion epidermis (θ2) (p < 0.05) (Figure 4A). Appressorium and infection hyphae formation were induced at 4 h and 6 h, respectively, on pear wax extract-coated onion epidermis (θ2) (Figure 4B,C). The appressorium formation rate of the ΔAaSlt2 strain on the pear wax extract-coated onion epidermis (θ2) was dramatically reduced by 75% at 6 h compared to the WT (p < 0.05) (Figure 4B). After 8 h of incubation, ΔAaSlt2 showed a 92% reduction in infection hyphae production on pear wax extract-coated onion epidermis (θ2) compared to WT (p < 0.05) (Figure 4C), indicating that AaSlt2 is important for infection structure formation in A. alternata when recognizing and responding to pear wax.

3.4. AaSlt2 Is Susceptible to Cell Wall Inhibitor Agents and Oxidative Stressors

Slt2, a crucial enzyme in MAPK pathways that responds to oxidative stress and cell wall integrity, has unknown mechanisms in A. alternata. As illustrated in Figure 5, in the presence of congo red, SDS, and H2O2, growth inhibition was more pronounced in ΔAaSlt2 compared to WT. The growth of ΔAaSlt2 was decreased by 56.38% when grown on PDA with 3 mM H2O2. With the addition of 100 μM congo red, the ΔAaSlt2 only formed colonies with limited growth compared to WT, and the inhibitory rates were 68.19%, indicating that AaSlt2 is involved in cell wall integrity and oxidative stress adaption in A. alternata.

3.5. AaSlt2 Is Essential for the Pathogenicity of A. alternata on Pear Fruit

To evaluate the role of AaSlt2 in the pathogenicity of A. alternata, 20 µL spore suspensions of WT, ΔAaSlt2, and ΔAaSlt2-C were inoculated into damaged pear fruit. The results indicated that WT, ΔAaSlt2, and ΔAaSlt2-C developed black spots on pears within 1–3 days; however, with extended incubation, the lesion diameter on pears infected with the ΔAaSlt2 strain exhibited minimal expansion (Figure 6A). The lesion diameters on pear fruit inoculated with the ΔAaSlt2 strain diminished by 57.07% after 3 days of inoculation compared to the WT (Figure 6B), demonstrating that AaSlt2 is essential for the pathogenicity of A. alternata.

3.6. Deletion of AaSlt2 Results in a Decrease of CWDEs Activity

To further investigate the impact of AaSlt2 on the pathogenicity of A. alternata, the activity of CWDEs (PG, PMG, Cx, and β-glucosidase) was assessed. As illustrated in Figure 7, the activities of PG, PMG, Cx, and β-glucosidase were diminished in the ΔAaSlt2 strain compared to the WT. PG activity increased following 1 day of inoculation, subsequently declining sharply, with the PG activity in the ΔAaSlt2 strain exhibiting a substantial reduction of 76.27% after 5 days of incubation (p < 0.05) in comparison to the WT (Figure 7A). The PMG activity of the ΔAaSlt2 strain decreased by 47.70% after 1 day of incubation compared to the WT (p < 0.05) (Figure 7B). Likewise, Cx and β-glucosidase activity diminished by 72.55% and 47.33% after 5 days of inoculation compared to WT, respectively (p < 0.05) (Figure 7C,D), suggesting that AaSlt2 influences the pathogenicity of A. alternata by modulating the activities of CWDEs.

3.7. Deletion of AaSlt2 Results in a Reduction of Melanin Accumulation and Toxin Production of A. alternata

To investigate the potential influence of AaSlt2 on the colony pigmentation of A. alternata, we investigated the accumulation of melanin. The melanin content of ΔAaSlt2 was diminished by 41.45% relative to that of the WT (p < 0.05) and was largely reinstated in the ΔAaSlt2-C (Figure 8A). ALT and TEN are significant toxins in A. alternata. The ALT and TEN levels of ΔAaSlt2 were reduced by 45.21% and 90.96%, respectively, compared to the WT (p < 0.05), respectively (Figure 8B). These results indicated that AaSlt2 regulated melanin accumulation and toxin biosynthesis in A. alternata.

4. Discussion

MAPK cascades are conserved signaling pathways in filamentous fungi, implicated in growth, secondary metabolism, stress adaptation, cell wall integrity, and virulence [32,33]. A considerable amount of research has been conducted to elucidate the mechanism of action of these conserved MAPK cascades in various fungi. BbSte12 and Bbmpk1 are implicated in the proliferation, oxidative stress response, and hyphal differentiation of B. bassiana [34]. Slt2-MAPK, a component of the MAPK cascade pathways, is crucial in regulating cell differentiation, cell wall integrity, conidial germination, and pathogenicity [35,36]. Slt2 is a crucial protein in the Slt2-MAPK pathway, which regulates cellular responses to host and environmental stimuli in fungal infections [37].
The AaSlt2 gene was identified from A. alternata JT-03 in our research. The homology study of amino acid sequences indicated that Slt2 exhibited a homology of 69.28% in A. alternata, S. cerevisiae, and Colletotrichum orchidophilum. Analysis of the amino acid sequence indicated that the protein encoded by AaSlt2 possesses the phosphorylation motif ‘TEY’ and the distinctive sequence ‘VlHRDLKPGNLLV’ (Figure 1). MAPK cascades are part of the Ser/Thr protein kinase family, characterized by a conserved threonine-x-tyrosine (TXY) motif across many fungi, suggesting that Slt2 has been highly conserved throughout evolution [38].
In this investigation, we described the AaSlt2 deletion mutant (ΔAaSlt2) and the complementary strain (ΔAaSlt2-C) derived from A. alternata JT-03. Phenotypic analysis indicated that the deletion of AaSlt2 influenced spore morphology, vegetative growth, and sporulation in A. alternata (Figure 2). In Mycosphaerella graminicola, the deletion of Slt2 resulted in considerable deficiencies in conidia [39]. These results are consistent with a prior publication on Arthrobotrys oligospora [40]. In Citrus A. alternata, the Slt2 deletion mutant exhibits significant impairments in colony proliferation, conidial germination, and aerial hyphal growth, with conidia sizes exceeding those of the wild type. Bashi et al. [41] revealed that the loss of smk3 greatly impacted sclerotium formation and aerial hyphal growth in Sclerotinia sclerotiorum, demonstrating that Slt2 is crucial for spore morphology, vegetative growth, and sporulation across several fungal species.
Fungal pathogens can detect and react to the physicochemical properties of plant surfaces, such as hydrophobicity and the composition of cutin and wax, subsequently activating G proteins, cAMP/PKA, and MAPK signaling pathways to modulate conidial development, appressorium formation, and pathogenicity [42,43]. Our prior research indicated that the Hog1-MAPK pathway is involved in the infection structure formation of A. alternata on hydrophobic and pear wax extract-coated surfaces [18,44]. However, the reaction of Slt2-MAPK to the pear wax of A. alternata needed additional clarification. Consequently, we assessed the gene expression level of the AaSlt2 gene on surfaces coated with hydrophobic and pear wax extract. The findings indicated that the expression level of AaSlt2 was markedly up-regulated during infection structure formation in A. alternata on a surface coated with pear wax extract (Figure 3). Moreover, experiments on onion epidermis indicated that the absence of AaSlt2 reduced spore germination, appressorium formation, and infection hyphae formation rates (Figure 4), implying that AaSlt2 is crucial for infection structure formation in A. alternata when identifying and reacting to pear wax. Likewise, in Magnaporthe oryzae [45], B. cinerea [46] and S. sclerotiorum [41], the loss of Slt2 impacted the production of infection structures.
The Slt2-MAPK pathway plays a conserved role in maintaining cell wall integrity. Congo red and SDS impede the polymerization reaction of chitin, thereby damaging the cell wall [47]. Various exogenous chemicals were introduced to the PDA for investigation. The results indicated that growth inhibition was more pronounced in ΔAaSlt2 compared with that in WT (Figure 5), suggesting that AaSlt2 plays a vital role in preserving cell wall integrity and adapting to oxidative stress in A. alternata, which exhibited similarities to prior research on G. lucidum [22]. Nonetheless, in B. cinerea, the absence of Slt2 did not influence protoplast release or cell wall integrity, suggesting that Slt2 serves distinct roles in maintaining cell wall integrity across various fungi. As a result, certain pathways may be elucidated using transcriptome and metabolomics research.
The Slt2-MAPK pathway also plays a conserved role in pathogenicity. Infection experiments demonstrated that the deletion of AaSlt2 markedly reduced pathogenicity (Figure 6). Similarly, the deletion of Slt2 markedly reduced pathogenicity in P. sojae [21], B. bassiana [48], and Cryphonectria parasitica [49]. Fungal virulence determinants comprise CWDEs, effector proteins, and toxins. Pathogens can secrete CWDEs to breakdown the host plant’s cell wall during infection. We assessed the activity of CWDEs. The findings indicated a considerable reduction in PG, PMG, Cx, and β-glucosidase activity in the ΔAaSlt2 strain (Figure 7).
Melanin included in the fungal cell wall safeguards fungal pathogens against environmental stressors. Nonetheless, melanin functioned as a harmful component in fungal pathogens [50]. A. alternata can synthesize melanin, which imparts color to its colonies. Upon inoculation on PDA, it first develops white mycelium, and the colony color transitions from grey to olive or olive-brown over time [51]. In this scenario, melanin accumulation was markedly reduced in the ΔAaSlt2 strain compared to the wild type (Figure 8A), consistent with prior research on Cochliobolus heterostrophus [52] and citrus A. alternata [19]. Melanin production, facilitated by the MAPK cascade pathway, is linked with virulence in pear A. alternata. Li et al. [53] established that the deletion of AaPKS1 decreased the pathogenicity of A. alternata.
During pathogenesis, several Alternaria species can produce both host-specific toxins (HSTs) and non-host specific toxins (NHST). The predominant category of toxins linked to A. alternata JT-03 comprises ALT and TEN. ALT and TEN are non-host specific toxins that result in numerous food safety issues and endanger human health. The elimination of AaSlt2 dramatically lowered ALT and TEN content (Figure 8B), suggesting that AaSlt2 is involved in the toxin synthesis process. In Fusarium graminearum, the ablation of Slt2 reduced toxin levels [54]. However, the precise processes of AaSlt2 in mycotoxin generation require additional investigation through the study of toxin-encoding genes by molecular methods.

5. Conclusions

In conclusion, we successfully engineered AaSlt2 mutant (ΔAaSlt2) and complementary strains (ΔAaSlt2-C), demonstrating that AaSlt2 regulates spore morphology, vegetative growth, and environmental stress adaptation in A. alternata. The current work has shown that AaSlt2 is essential to infection structure formation and the virulence of A. alternata. The findings enhance our understanding of the molecular mechanisms of A. alternata infection and establish a theoretical foundation for the development of target-specific fungicides for postharvest disease management.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof10110774/s1, Figure S1: Strategy of homologous recombination knockout of AaSlt2. The target fragment was recovered through a hph (Hygromycin B) resistance gene cassette; Figure S2: Verification of positive transformant. (A) Validation of AaSlt2 full-length. (B) Validation of targeted insertion. (C) Strategy of full-length verification of AaSlt2 gene. (D) Strategy of targeted insertion verification; Figure S3: AaSlt2 gene expression analysis. Note: Bars indicate standard error (±SE). Different letters indicate significant differences (p < 0.05); Figure S4: Amplification of AaSlt2 cDNA fragment for complementation. Figure S5: Conserved domain of AaSlt2; Table S1: Primers used for amplification of AaSlt2-up and AaSlt2-down; Table S2: Primers used for PCR amplification and qPCR in positive transformant; Table S3: Primers used for amplification of AaSlt2 cDNA; Table S4: Primers used for PCR amplification in complementation transformants; Table S5: Primers used for qRT-PCR. GAPDH was used as the reference gene.

Author Contributions

Conceptualization, Q.J.; methodology, T.W.; software, Q.J. and M.Z.; formal analysis, T.W. and Y.L.; investigation, T.W. and M.Z.; resources, Y.L., Q.J., T.W. and Y.B.; data curation, Q.J. and X.W.; writing—original draft preparation, Q.J.; writing—review and editing, Q.J., T.W., Y.L. and D.B.P.; supervision, Y.L., Y.B. and D.B.P.; project administration, Y.L. and X.W.; funding acquisition, Y.L. and X.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China, grant number 32060567, 32372411 and Youth Science and Technology Fund of Gansu Province, grant number 22JR5RA879.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

Acknowledgments

The authors would like to thank anonymous reviewers for helpful comments.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Pan, T.T.; Chyngyz, E.; Sun, D.W.; Paliwal, J.; Pu, H.B. Pathogenetic process monitoring and early detection of pear black spot disease caused by Alternaria alternata using hyperspectral imaging. Postharvest Biol. Technol. 2019, 154, 96–104. [Google Scholar] [CrossRef]
  2. Zhang, S.J.; Wang, Q.; Guo, Y.J.; Kang, L.; Yu, Y.W. Carbon monoxide enhances the resistance of jujube fruit against postharvest Alternaria rot. Postharvest Biol. Technol. 2020, 101, 1040. [Google Scholar] [CrossRef]
  3. Ahmad, T.; Liu, Y.; Huang, S.J.; Moosa, A. First record of Alternaria alternata causing postharvest fruit rot of sweet cherry (Prunus avium) in China. Plant Dis. 2020, 104, 2030. [Google Scholar] [CrossRef]
  4. Wang, Q.; Cai, R.; Wang, Z.; Yue, T.; Cui, L. Advances in detection and control of Alternaria toxin in fruits and vegetables. J. Food Saf. Food Qual. 2020, 11, 114–120. [Google Scholar] [CrossRef]
  5. Wang, F.; Saito, S.; Michailides, T.; Xiao, C. Fungicide resistance in Alternaria alternata from blueberry in California and its impact on control of Alternaria rot. Plant Dis. 2022, 106, 1446–1453. [Google Scholar] [CrossRef]
  6. Xu, H.; Xu, X.; Wang, Y.; Bajpai, V.; Huang, L.; Chen, Y.; Baek, K. The mitogen-activated protein kinase signal transduction pathways in Alternaria species. Plant Pathol. J. 2012, 28, 227–238. [Google Scholar] [CrossRef]
  7. Li, S.; Han, X.; Lu, Z.; Qiu, W.; Yu, M.; Li, H.; He, Z.; Zhuo, R. MAPK cascades and transcriptional factors: Regulation of heavy metal tolerance in plants. Int. J. Mol. Sci. 2022, 23, 4463. [Google Scholar] [CrossRef]
  8. Yu, L.; Xiong, D.G.; Han, Z.; Liang, Y.M.; Tian, C.M. The mitogen-activated protein kinase gene CcPmk1 is required for fungal growth, cell wall integrity and pathogenicity in Cytospora chrysosperma. Fungal Genet. Biol. 2019, 128, 1–13. [Google Scholar] [CrossRef]
  9. Cong, J.; Xiao, K.Q.; Jiao, W.L.; Zhang, C.; Zhang, X.H.; Liu, J.L.; Zhang, Y.H.; Pan, H.Y. The coupling between cell wall integrity mediated by MAPK kinases and SsFkh1 is involved in sclerotia formation and pathogenicity of Sclerotinia sclerotiorum. Front. Microbiol. 2022, 13, 816091. [Google Scholar] [CrossRef]
  10. Cao, H.; Gong, H.; Song, T.; Yu, M.; Pan, X.; Yu, J.; Qi, Z.; Du, Y.; Liu, Y. The adaptor protein UvSte50 governs fungal pathogenicity of Ustilaginoidea virens via the MAPK signaling pathway. J. Fungi 2022, 8, 954. [Google Scholar] [CrossRef]
  11. He, P.; Wang, Y.; Wang, X.; Zhang, X.; Tian, C. The mitogen-activated protein kinase CgMK1 governs appressorium formation, melanin synthesis, and plant infection of Colletotrichum gloeosporioides. Front. Microbiol. 2017, 8, 2216. [Google Scholar] [CrossRef] [PubMed]
  12. Molina, M.; Cid, V.; Martín, H. Fine regulation of Saccharomyces cerevisiae MAPK pathways by post-translational modifications. Yeast 2010, 27, 503–511. [Google Scholar] [CrossRef] [PubMed]
  13. Jin, K.; Han, L.; Xia, Y. MaMk1, a FUS3/KSS1-type mitogen-activated protein kinase gene, is required for appressorium formation, and insect cuticle penetration of the entomopathogenic fungus Metarhizium acridum. J. Invertebr. Pathol. 2014, 115, 68–75. [Google Scholar] [CrossRef] [PubMed]
  14. Qu, Y.; Wang, J.; Huang, P.; Liu, X.; Lu, J.; Lin, F. PoRal2 is involved in appressorium formation and virulence via Pmk1 MAPK pathways in the rice blast fungus Pyricularia oryzae. Front. Plant Sci. 2021, 12, 702368. [Google Scholar] [CrossRef] [PubMed]
  15. Li, C.; Sun, W.; Cao, S.; Hou, R.; Li, X.; Ming, L.; Kan, J.; Zhao, Y.; Liu, F. The CfMK1 gene regulates reproduction, appressorium formation, and pathogenesis in a pear anthracnose-causing fungus. J. Fungi 2022, 8, 77. [Google Scholar] [CrossRef]
  16. Yin, Z.Q.; Bi, W.; Mi, Q.L.; Kang, Z.T.; Liu, C.J.; Yang, J.K.; Luo, Y.Y. Conserved and divergent roles of the HOG1 kinase of Alternaria longipes in mycelial and conidial development, multi-stress responses, melanin production and pathogenicity. Eur. J. Plant Pathol. 2016, 147, 415–430. [Google Scholar] [CrossRef]
  17. Zheng, D.W.; Wang, Y.; Han, Y.; Xu, J.R.; Wang, C.F. UvHOG1 is important for hyphal growth and stress responses in the rice false smut fungus Ustilaginoidea virens. Sci. Rep. 2016, 6, 24824. [Google Scholar] [CrossRef]
  18. Zhang, M.; Wang, T.L.; Li, Y.C.; Bi, Y.; Li, R.; Yuan, J.; Xu, W.Y.; Prusky, D.B. AaHog1 regulates infective structural differentiation mediated by physicochemical signals from pear fruit cuticular wax, stress response, and Alternaria alternata pathogenicity. J. Fungi 2022, 8, 266. [Google Scholar] [CrossRef]
  19. Yago, J.I.; Lin, C.H.; Chung, K.R. The SLT2 mitogenactivated protein kinase-mediated signaling pathway governs conidiation, morphogenesis, fungal virulence and production of toxin and melanin in the tangerine pathotype of Alternaria alternata. Mol. Plant Pathol. 2011, 12, 653–665. [Google Scholar] [CrossRef]
  20. Luo, X.; Keyhani, N.; Yu, X.; He, Z.; Luo, Z.; Pei, Y.; Zhang, Y. The MAP kinase Bbslt2 controls growth, conidiation, cell wall integrity, and virulence in the insect pathogenic fungus Beauveria bassiana. Fungal Genet. Biol. 2012, 49, 544–555. [Google Scholar] [CrossRef]
  21. Li, A.; Zhang, M.; Wang, Y.; Li, D.; Liu, X.; Tao, K.; Ye, W.; Wang, Y. PsMPK1, an SLT2-type mitogen-activated protein kinase, is required for hyphal growth, zoosporogenesis, cell wall integrity, and pathogenicity in Phytophthora sojae. Fungal Genet. Biol. 2014, 65, 14–24. [Google Scholar] [CrossRef] [PubMed]
  22. Chen, D.D.; Shi, L.; Yue, S.N.; Zhang, T.J.; Wang, S.L.; Liu, Y.N.; Ren, A.; Zhu, J.; Yu, H.S.; Zhao, M.W. The Slt2-MAPK pathway is involved in the mechanism by which target of rapamycin regulates cell wall components in Ganoderma lucidum. Fungal Genet. Biol. 2019, 123, 70–77. [Google Scholar] [CrossRef] [PubMed]
  23. Zhang, F.; Huang, L.; Deng, J.; Tan, C.; Geng, L.; Liao, Y.; Yuan, J.; Wang, S. A cell wall integrity-related MAP kinase kinase kinase AflBck1 is required for growth and virulence in fungus Aspergillus flavus. Mol. Plant-Microbe Interact. 2020, 33, 680–692. [Google Scholar] [CrossRef] [PubMed]
  24. Zhang, F.; Geng, L.P.; Deng, J.L.; Huang, L.H.; Zhong, H.; Xin, S.J.; Fasoyin, O.E.; Wang, S.H. The MAP kinase AflSlt2 modulates aflatoxin biosynthesis and peanut infection in the fungus Aspergillus flavus. Int. J. Food Microbiol. 2020, 322, 108576. [Google Scholar] [CrossRef]
  25. Spada, M.; Pugliesi, C.; Fambrini, M.; Pecchia, S. Silencing of the Slt2-type MAP kinase Bmp3 in Botrytis cinerea by application of exogenous dsRNA affects fungal growth and virulence on Lactuca sativa. Int. J. Mol. Sci. 2021, 22, 5362. [Google Scholar] [CrossRef]
  26. Wang, T.L.; Zhang, M.; Li, Y.C.; Bi, Y.; Zhang, T.T.; Zheng, X.Y. MAPK signal cascade pathway is involved in sening and responding process of Alternaria alternata to cues from pear fruit surface. J. Gansu Agric. Univ. 2019, 54, 159–168+174. [Google Scholar] [CrossRef]
  27. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
  28. Tang, Y.; Li, Y.C.; Bi, Y.; Wang, Y. Role of pear fruit cuticular wax and surface hydrophobicity in regulating the prepenetration phase of Alternaria alternata infection. J. Phytopathol. 2017, 165, 313–322. [Google Scholar] [CrossRef]
  29. Jia, M.; Liu, X.T.; Zhao, H.; Ni, Y.X.; Liu, H.Y.; Tian, B.M. Cell-wall-degrading enzymes produced by sesame leaf spot pathogen Corynespora cassiicola. J. Phytopathol. 2020, 169, 186–192. [Google Scholar] [CrossRef]
  30. Zhang, M.; Li, Y.C.; Wang, T.L.; Bi, Y.; Li, R.; Huang, Y.; Mao, R.Y.; Jiang, Q.Q.; Liu, Y.X.; Prusky, D.B. AaPKAc regulates differentiation of infection structures induced by physicochemical signals from pear fruit cuticular wax, secondary metabolism, and pathogenicity of Alternaria alternata. Front. Plant Sci. 2021, 12, 642601. [Google Scholar] [CrossRef]
  31. Xu, L.; Tao, N.; Yang, W.; Jing, G. Cinnamaldehyde damaged the cell membrane of Alternaria alternata and induced the degradation of mycotoxins in vivo. Ind. Crop. Prod. 2018, 112, 427–433. [Google Scholar] [CrossRef]
  32. Chen, X.X.; Xu, C.; Qian, Y.; Liu, R.; Zhang, Q.Q.; Zeng, G.H.; Zhang, X.; Zhao, H.; Fang, W.G. MAPK cascade-mediated regulation of pathogenicity, conidiation and tolerance to abiotic stresses in the entomopathogenic fungus Metarhizium robertsii. Environ. Microbiol. 2016, 18, 1048–1062. [Google Scholar] [CrossRef] [PubMed]
  33. Osés-Ruiz, M.; Cruz-Mireles, N.; Martin-Urdiroz, M.; Soanes, D.M.; Eseola, A.B.; Tang, B.; Derbyshire, P.; Nielsen, M.; Cheema, J.; Were, V.; et al. Appressorium-mediated plant infection by Magnaporthe oryzae is regulated by a Pmk1-dependent hierarchical transcriptional network. Nat. Microbiol. 2021, 6, 1383–1397. [Google Scholar] [CrossRef] [PubMed]
  34. Zhao, X.; Jiang, Y.H.; Wang, H.F.; Lu, Z.Y.; Huang, S.S.; Luo, Z.B.; Zhang, L.Y.; Lv, T.; Tang, X.H.; Zhang, Y.J. Fus3/Kss1-MAP kinase and Ste12-like control distinct biocontrol-traits besides regulation of insect cuticle penetration via phosphorylation cascade in a filamentous fungal pathogen. Pest Manag. Sci. 2023, 79, 2611–2624. [Google Scholar] [CrossRef] [PubMed]
  35. Martín, H.; Rodríguez-Pachón, J.M.; Ruiz, C.; Nombela, C.; Molina, M. Regulatory mechanisms for modulation of signaling through the cell integrity Slt2-mediated pathway in Saccharomyces cerevisiae. J. Biol. Chem. 2000, 275, 1511–1519. [Google Scholar] [CrossRef]
  36. Turrà, D.; Segorbe, D.; Pietro, A.D. Protein kinases in plant-pathogenic fungi: Conserved regulators of infection. Annu. Rev. Phytopathol. 2014, 52, 267–288. [Google Scholar] [CrossRef]
  37. Leng, G.; Song, K. Direct interaction of Ste11 and Mkk1/2 through Nst1 integrates high-osmolarity glycerol and pheromone pathways to the cell wall integrity MAPK pathway. FEBS Lett. 2016, 590, 148–160. [Google Scholar] [CrossRef]
  38. Jiang, C.; Zhang, X.; Liu, H.Q.; Xu, J.R. Mitogen-activated protein kinase signaling in plant pathogenic fungi. PLoS Pathog. 2018, 14, e1006875. [Google Scholar] [CrossRef]
  39. Mehrabi, R.; Van, D.; Lee, T.; Waalwijk, C.; Kema, G.H.J. MgSlt2, a cellular integrity MAP kinase gene of the fungal wheat pathogen Mycosphaerella graminicola, is dispensable for penetration but essential for invasive growth. Mol. Plant-Microbe Interact. 2006, 19, 389–398. [Google Scholar] [CrossRef]
  40. Zhen, Z.; Xing, X.; Xie, M.; Yang, L.; Yang, X.; Zheng, Y.; Chen, Y.; Ma, N.; Li, Q.; Zhang, K.; et al. MAP kinase Slt2 orthologs play similar roles in conidiation, trap formation, and pathogenicity in two nematode-trapping fungi. Fungal Genet. Biol. 2018, 116, 42–50. [Google Scholar] [CrossRef]
  41. Bashi, Z.D.; Gyawali, S.; Bekkaoui, D.; Coutu, C.; Lee, L.; Poon, J.; Rimmer, S.R.; Khachatourians, G.G.; Hegedus, D.D. The Sclerotinia sclerotiorum Slt2 mitogen-activated protein kinase ortholog, SMK3, is required for infection initiation but not lesion expansion. Can. J. Microbiol. 2016, 62, 836–850. [Google Scholar] [CrossRef] [PubMed]
  42. Kou, Y.; Naqvi, N.I. Surface sensing and signaling networks in plant pathogenic fungi. Semin. Cell Dev. Biol. 2016, 57, 84–92. [Google Scholar] [CrossRef] [PubMed]
  43. Santos, I.B.D.; Lopes, M.D.S.; Bini, A.P.; Tschoeke, B.A.P.; Verssani, B.A.W.; Figureueredo, E.F.; Cataldi, T.R.; Marques, J.P.R.; Silva, L.D.; Labate, C.A.; et al. The eucalyptus cuticular waxes contribute in preformed defense against Austropuccinia psidii. Front. Plant Sci. 2019, 9, 1978. [Google Scholar] [CrossRef] [PubMed]
  44. Yuan, J.; Liu, Y.X.; Li, Y.C.; Zong, Y.Y.; Bi, Y.; Prusky, D.B. The sensor protein AaSln1 is involved in differentiation of infection structures, osmotic stress tolerance and virulence in Alternaria alternata. Postharvest Biol. Technol. 2024, 209, 112697. [Google Scholar] [CrossRef]
  45. Liu, W.; Zhou, X.; Li, G.; Li, L.; Kong, L.; Wang, C.; Zhang, H.; Xu, J. Multiple plant surface signals are sensed by different mechanisms in the rice blast fungus for appressorium formation. PLoS Pathog. 2011, 7, e1001261. [Google Scholar] [CrossRef]
  46. Rui, O.; Hahn, M. The Slt2-type MAP kinase Bmp3 of Botrytis cinerea is required for normal saprotrophic growth, conidiation, plant surface sensing and host tissue colonization. Mol. Plant Pathol. 2007, 8, 173–184. [Google Scholar] [CrossRef]
  47. Schroeder, L.; Ikui, A.E. Tryptophan confers resistance to SDS-associated cell membrane stress in Saccharomyces cerevisiae. PLoS ONE 2019, 14, e0199484. [Google Scholar] [CrossRef]
  48. Huang, S.; He, Z.; Zhang, S.; Keyhani, N.; Song, Y.; Yang, Z.; Jiang, Y.; Zhang, W.; Pei, Y.; Zhang, Y. Interplay between calcineurin and the Slt2 MAP-kinase in mediating cell wall integrity, conidiation and virulence in the insect fungal pathogen Beauveria bassiana. Fungal Genet. Biol. 2015, 83, 78–91. [Google Scholar] [CrossRef]
  49. So, K.K.; Ko, Y.H.; Chun, T.; Kim, J.M.; Kim, D.H. Mutation of the Slt2 ortholog from Cryphonectria parasitica results in abnormal cell wall integrity and sectorization with impaired pathogenicity. Sci. Rep. 2017, 7, 9038. [Google Scholar] [CrossRef]
  50. Jacobson, E.S. Pathogenic roles for fungal melanins. Clin. Microbiol. Rev. 2000, 13, 708–717. [Google Scholar] [CrossRef]
  51. Kustrzeba-Wójcicka, I.; Siwak, E.; Terlecki, G.; Wolańczyk-Mędrala, A.; Mędrala, W. Alternaria alternata and its allergens: A comprehensive review. Clin. Rev. Allergy Immunol. 2014, 47, 354–365. [Google Scholar] [CrossRef] [PubMed]
  52. Eliahu, N.; Igbaria, A.; Rose, M.S.; Horwitz, B.A.; Lev, S. Melanin biosynthesis in the maize pathogen Cochliobolus heterostrophus depends on two mitogen-activated protein kinases, CHk1 and MPs1, and the transcription factor cmr1. Eukaryotic Cell 2007, 6, 421–429. [Google Scholar] [CrossRef] [PubMed]
  53. Li, R.; Xu, W.Y.; Zong, Y.Y.; Wang, X.J.; Li, Y.C.; Bi, Y.; Prusky, D.B. Melanin synthesis gene Aapks contributes to appressorium formation, stress response, cell well integrity and virulence in Alternaria alternata. Postharvest Biol. Technol. 2023, 198, 112247. [Google Scholar] [CrossRef]
  54. Hou, Z.; Xue, C.; Peng, Y.; Katan, T.; Kistler, H.C.; Xu, J.R. A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility, heterokaryon formation, and plant infection. Mol. Plant-Microbe Interact. 2002, 15, 1119–1127. [Google Scholar] [CrossRef]
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Figure 1. Amino acid sequence alignments of AaSlt2 with homologous protein sequences of Saccharomyces cerevisiae (NP_011895.1) and Colletotrichum orchidophilum (XP_022474739). The orange box indicates the phosphorylation motif and the blue box indicates the characteristic sequences.
Figure 1. Amino acid sequence alignments of AaSlt2 with homologous protein sequences of Saccharomyces cerevisiae (NP_011895.1) and Colletotrichum orchidophilum (XP_022474739). The orange box indicates the phosphorylation motif and the blue box indicates the characteristic sequences.
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Figure 2. AaSlt2 is required for vegetative growth and spore development of A. alternata. (A) Spore development of the indicated strains on PDA medium. The black arrow indicates the abnormal spore of A. alternata. (B) 5-day-old PDA medium and colony morphology of the WT, ΔAaSlt2, and ΔAaSlt2-C at 28 °C. (C) Colony diameter of WT, ΔAaSlt2, and ΔAaSlt2-C on PDA medium 5 days after incubation at 28 °C. (D) Sporulation of WT, ΔAaSlt2, and ΔAaSlt2-C on PDA medium 5 days after incubation at 28 °C. Bars indicate standard error (±SE). Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
Figure 2. AaSlt2 is required for vegetative growth and spore development of A. alternata. (A) Spore development of the indicated strains on PDA medium. The black arrow indicates the abnormal spore of A. alternata. (B) 5-day-old PDA medium and colony morphology of the WT, ΔAaSlt2, and ΔAaSlt2-C at 28 °C. (C) Colony diameter of WT, ΔAaSlt2, and ΔAaSlt2-C on PDA medium 5 days after incubation at 28 °C. (D) Sporulation of WT, ΔAaSlt2, and ΔAaSlt2-C on PDA medium 5 days after incubation at 28 °C. Bars indicate standard error (±SE). Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
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Figure 3. Relative expression levels of AaSlt2 during infection structural differentiation of A. alternata on hydrophobic and pear wax extract-coated surface. Intra-group comparisons were made to compare the changes in AaSlt2 gene expression during infection structural differentiation of A. alternata under hydrophobic and pear wax substrates, respectively. Extra-group were made to compare the changes in AaSlt2 gene expression under hydrophobic and pear wax substrates at the same stage. Vertical lines indicate the standard error (±SE) of the means. Capital letters indicate intra-group differences. Lowercase letters indicate extra-group differences. Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
Figure 3. Relative expression levels of AaSlt2 during infection structural differentiation of A. alternata on hydrophobic and pear wax extract-coated surface. Intra-group comparisons were made to compare the changes in AaSlt2 gene expression during infection structural differentiation of A. alternata under hydrophobic and pear wax substrates, respectively. Extra-group were made to compare the changes in AaSlt2 gene expression under hydrophobic and pear wax substrates at the same stage. Vertical lines indicate the standard error (±SE) of the means. Capital letters indicate intra-group differences. Lowercase letters indicate extra-group differences. Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
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Figure 4. AaSlt2 regulates the infection structure differentiation of A. alternata in vivo. WT, ΔAaSlt2, and ΔAaSlt2-C spore suspensions (20 µL) were dripped onto intact onion epidermis (θ1) and pear wax extract-coated onion epidermis (θ2) with three replicates and incubated at 28 °C. The percentages of spore germination, appressorium formation, and infection hyphae formation were calculated under a microscope at 2, 4, 6 and 8 h after incubation. The spore germination rate, appressorium formation rate, and infection hyphae formation rate were obtained by counting the number of germinated spores, appressorium formation, and infection hyphae formation in 100 spores and multiplying the result by 100%. (A) The spore germination rate of A. alternata. (B) The appressorium formation rate of A. alternata. (C) The infection hyphae formation rate of A. alternata. Vertical lines indicate standard error (±SE). Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
Figure 4. AaSlt2 regulates the infection structure differentiation of A. alternata in vivo. WT, ΔAaSlt2, and ΔAaSlt2-C spore suspensions (20 µL) were dripped onto intact onion epidermis (θ1) and pear wax extract-coated onion epidermis (θ2) with three replicates and incubated at 28 °C. The percentages of spore germination, appressorium formation, and infection hyphae formation were calculated under a microscope at 2, 4, 6 and 8 h after incubation. The spore germination rate, appressorium formation rate, and infection hyphae formation rate were obtained by counting the number of germinated spores, appressorium formation, and infection hyphae formation in 100 spores and multiplying the result by 100%. (A) The spore germination rate of A. alternata. (B) The appressorium formation rate of A. alternata. (C) The infection hyphae formation rate of A. alternata. Vertical lines indicate standard error (±SE). Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
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Figure 5. Colony morphology (A) and inhibition of growth (B) of WT, ΔAaSlt2, and ΔAaSlt2-C strains on PDA with different exogenous compounds. Lesions were measured at 5 days. Inhibition of growth (%) = (x − y)/x, where x is the colony diameter of WT, ΔAaSlt2 and ΔAaSlt2-C strains on PDA, y is the colony diameter of WT, ΔAaSlt2 and ΔAaSlt2-C strains on PDA with different exogenous compounds. Means and standard deviations were calculated from three replicates. Vertical lines indicate standard error (±SE) of the means. Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
Figure 5. Colony morphology (A) and inhibition of growth (B) of WT, ΔAaSlt2, and ΔAaSlt2-C strains on PDA with different exogenous compounds. Lesions were measured at 5 days. Inhibition of growth (%) = (x − y)/x, where x is the colony diameter of WT, ΔAaSlt2 and ΔAaSlt2-C strains on PDA, y is the colony diameter of WT, ΔAaSlt2 and ΔAaSlt2-C strains on PDA with different exogenous compounds. Means and standard deviations were calculated from three replicates. Vertical lines indicate standard error (±SE) of the means. Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
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Figure 6. The lesion morphology (A) and disease diameter (B). Lesions were measured at 3, 5, 7, 9 and 11 days, respectively. Means and standard deviations were calculated from three replicates. Vertical lines indicate standard error (±SE) of the means. Intra-group comparisons were made to compare changes in the same treatment (inoculation with WT/ΔAaSlt2/ΔAaSlt2-C) at different incubation times (3, 5, 7, 9 and 11 days). Extra-group were made to compare the changes of differences treatments (inoculation with WT, ΔAaSlt2 and ΔAaSlt2-C) at the same incubation times. Capital letters indicate intra-group differences. Lowercase letters indicate extra-group differences. Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
Figure 6. The lesion morphology (A) and disease diameter (B). Lesions were measured at 3, 5, 7, 9 and 11 days, respectively. Means and standard deviations were calculated from three replicates. Vertical lines indicate standard error (±SE) of the means. Intra-group comparisons were made to compare changes in the same treatment (inoculation with WT/ΔAaSlt2/ΔAaSlt2-C) at different incubation times (3, 5, 7, 9 and 11 days). Extra-group were made to compare the changes of differences treatments (inoculation with WT, ΔAaSlt2 and ΔAaSlt2-C) at the same incubation times. Capital letters indicate intra-group differences. Lowercase letters indicate extra-group differences. Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
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Figure 7. PG (A), PMG (B), Cx (C) and β-glucosidase (D) activity of WT, ΔAaSlt2 and ΔAaSlt2-C. Bars indicate standard error (±SE). Intra-group comparisons were made to compare changes in the same treatment (inoculation with WT/ΔAaSlt2/ΔAaSlt2-C) at different incubation times (3, 5, 7, 9 and 11 days). Extra-group were made to compare the changes of differences treatments (inoculation with WT, ΔAaSlt2 and ΔAaSlt2-C) at the same incubation times. Capital letters indicate intra-group differences. Lowercase letters indicate extra-group differences. Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
Figure 7. PG (A), PMG (B), Cx (C) and β-glucosidase (D) activity of WT, ΔAaSlt2 and ΔAaSlt2-C. Bars indicate standard error (±SE). Intra-group comparisons were made to compare changes in the same treatment (inoculation with WT/ΔAaSlt2/ΔAaSlt2-C) at different incubation times (3, 5, 7, 9 and 11 days). Extra-group were made to compare the changes of differences treatments (inoculation with WT, ΔAaSlt2 and ΔAaSlt2-C) at the same incubation times. Capital letters indicate intra-group differences. Lowercase letters indicate extra-group differences. Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
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Figure 8. A. alternata melanin content (A) and toxin content (B). Bars indicate standard error (±SE) of three replicates. Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
Figure 8. A. alternata melanin content (A) and toxin content (B). Bars indicate standard error (±SE) of three replicates. Different letters on the bars for each treatment indicate significant differences at p < 0.05 by Duncan’s multiple range test.
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MDPI and ACS Style

Jiang, Q.; Wang, T.; Li, Y.; Bi, Y.; Zhang, M.; Wang, X.; Prusky, D.B. AaSlt2 Is Required for Vegetative Growth, Stress Adaption, Infection Structure Formation, and Virulence in Alternaria alternata. J. Fungi 2024, 10, 774. https://doi.org/10.3390/jof10110774

AMA Style

Jiang Q, Wang T, Li Y, Bi Y, Zhang M, Wang X, Prusky DB. AaSlt2 Is Required for Vegetative Growth, Stress Adaption, Infection Structure Formation, and Virulence in Alternaria alternata. Journal of Fungi. 2024; 10(11):774. https://doi.org/10.3390/jof10110774

Chicago/Turabian Style

Jiang, Qianqian, Tiaolan Wang, Yongcai Li, Yang Bi, Miao Zhang, Xiaojing Wang, and Dov B. Prusky. 2024. "AaSlt2 Is Required for Vegetative Growth, Stress Adaption, Infection Structure Formation, and Virulence in Alternaria alternata" Journal of Fungi 10, no. 11: 774. https://doi.org/10.3390/jof10110774

APA Style

Jiang, Q., Wang, T., Li, Y., Bi, Y., Zhang, M., Wang, X., & Prusky, D. B. (2024). AaSlt2 Is Required for Vegetative Growth, Stress Adaption, Infection Structure Formation, and Virulence in Alternaria alternata. Journal of Fungi, 10(11), 774. https://doi.org/10.3390/jof10110774

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